Novel saitohin gene and uses of same

ABSTRACT

The present invention provides an isolated nucleic acid sequence encoding saitohin (STH), an isolated nucleic acid sequence that hybridizes to said sequence, and a purified protein encoded by said nucleic acid sequences. The present invention also provides a purified STH protein, and a method of making STH protein. The present invention is further directed to an antibody specific for STH, and a method for producing said antibody. Additionally, the present invention discloses a vector comprising a nucleic acid sequence encoding STH, a host cell transformed with said vector, and transgenic nonhuman animals. The present invention further provides methods for determining whether a subject has, or is at increased risk for developing, a neurodegenerative disease, and for assessing said subject&#39;s prognosis. Finally, the present invention discloses kits for determining whether a subject has, or is at increased risk for developing, a neurodegenerative disease.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIMH Grant No.38623. As such, the United States government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disease characterized bya progressive, inexorable loss of cognitive function (1). AD is the mostcommon late-onset dementia, affecting several million people in thedeveloped countries of the world. Approximately 4 million Americanssuffer from Alzheimer's disease, at an annual cost of about $100billion, making AD the third most costly disorder of aging. The diseaseis about twice as common in women as in men, and accounts for more than65% of the dementias in the elderly. Early identification is critical inprogressive conditions such as AD, because earlier treatment may be moreeffective than later treatment in preserving cognitive function.Furthermore, early detection may allow time to explore options fortreatment and care. To date, however, a cure for Alzheimer's disease isnot available, and cognitive decline is inevitable.

AD has two major neuropathological hallmarks: extracellular aggregates,called amyloid plaques, neuritic plaques, or senile plaques, which arecomposed of neurites, astrocytes, and glial cells around an amyloidcore, and which are located in the cerebral cortex; and intracellularaggregates, called neurofibrillary tangles, which are composed of pairedhelical filaments). While senile plaques and neurofibrillary tanglesoccur with normal aging, they are much more prevalent in persons withAlzheimer's disease. Although the processes of AD could be triggered bymany environmental insults, genetic studies have shown that mutationsand polymorphisms of particular genes can confer susceptibility to thisdegenerative process.

Genetic studies of Alzheimer's disease patients have identified severalearly onset (<65 years old) disease risk factors (e.g., mutations in theamyloid precursor protein, Presenilin 1 and Presenilin 2), and alate-onset (>65 years old) disease risk factor, the E4 allele ofapolipoprotein E (ApoE4) (see, e.g., U.S. Pat. Nos. 5,716,828;5,767,248; and 6,136,530). In the late-onset AD (LOAD) population, only50% of the subjects have been shown to carry the ApoE4 allele, which iscompelling evidence to support the existence of additional genetic riskfactors associated with AD. Indeed, recent linkage studies ofLOAD-affected sibling pairs have identified loci on chromosomes 9 and 10that may harbor risk-factor genes (2).

To date, the known risk factors for AD have been shown to modulateamyloid production or deposition; yet, none is necessary or sufficientfor the diagnosis of AD, and none has demonstrated a role in theformation of neurofibrillary tangles (NFT). Extensive research on NFThas been undertaken in conjunction with studies on the conversion oftau, a protein that is normally soluble, into a hyperphosphorylatedinsoluble protein that is detected in NFT. Tau, a microtubule-associateprotein is important in establishing and maintaining neuronalmorphology. In addition to its role in normal cells, tau protein isinvolved in many neurodegenerative diseases, including AD, as the maincomponent of intraneuronal aggregates. Some of the more common diseaseswith tau pathology include frontotemporal dementia (FTD), Pick'sdisease, and progressive supranuclear palsy (PSP) (3). The involvementof tau in these disorders has prompted much investigation into itsfunction and role in the progression of these disorders.

Recently, several tau-coding mutations have been shown to segregate withFTD, and there have been some intriguing results with transgenic miceexpressing the FTD-tau mutation, P301L, that develop tau aggregates (3,4). However, in AD, no mutations have been found in the tau gene,suggesting that other factors are likely involved in the formation oftau aggregates. These factors could have implications for otherneurodegenerative disorders with tau pathology (5-9).

In FTD and PSP subjects, recent genetic studies of the tau locus haveshown that several mutations and a polymorphism segregate with thesedisorders, respectively (3). The identification and investigation ofneighboring genes in the tau locus could be valuable in the study ofmolecular genetic risk factors in PSP, since there have been no reportedtau coding or intronic mutations (10). This information could also beuseful, not only for PSP, but for other neurodegenerative diseases, suchas AD. The identification of new risk factors associated with AD andother neurodegenerative diseases may assist in the diagnosis of suchdiseases facilitate future preventive and therapeutic measures directedto these diseases.

SUMMARY OF THE INVENTION

The present invention is predicated on the identification of a novelgene, saitohin (STH), within the tau locus, and the observation that thesaitohin-R (STH-R) allele of the STH gene is associated with anincreased risk of developing Alzheimer's disease (“AD”). On the basis ofthese findings, it is an object of the present invention to provide anisolated nucleic acid sequence encoding saitohin (STH), including boththe STH-Q and STH-R alleles, and an isolated nucleic acid sequence thathybridizes under high stringency conditions to a second nucleic acidthat is complementary to a nucleic acid sequence encoding STH.

The present invention also discloses a purified saitohin (STH) protein,including both the STH-Q and STH-R isoforms, and a purified proteinencoded by a nucleic acid sequence that hybridizes under high stringencyconditions to a second nucleic acid sequence that is complementary to anucleic acid sequence encoding STH, including both the STH-Q and STH-Ralleles. Also provided is a method of making STH protein, including boththe STH-Q and STH-R isoforms.

The present invention is further directed to an antibody specific forsaitohin (STH) protein, including both the STH-Q and STH-R isoforms, anda method for producing an antibody specific for STH protein, includingboth the STH-Q and STH-R isoforms.

Additionally, the present invention discloses a vector comprising anucleic acid sequence encoding saitohin (STH), including both the STH-Qand STH-R alleles, and a host cell transformed with a vector comprisinga nucleic acid sequence encoding STH, including both the STH-Q and STH-Ralleles.

The present invention is also directed to a transgenic non-human animalwhose genome comprises a disruption in its endogenous STH gene, and atransgenic non-human animal that overexpresses saitohin (STH) protein,including both the STH-Q and STH-R isoforms.

The present invention further provides a method for determining whethera subject has a neurodegenerative disease or is at increased risk fordeveloping neurodegenerative disease, comprising assaying a diagnosticsample of the subject for the presence of one or more alleles ofsaitohin (STH), wherein detection of the presence of STH-R allele isindicative that the subject has, or is at increased risk for developing,a neurodegenerative disease.

The present invention also provides a method for assessing the prognosisof a subject who has, or may develop, a neurodegenerative disease,comprising assaying a diagnostic sample of the subject for the presenceof one or more alleles of saitohin (STH), wherein the presence of twoSTH-R alleles in the diagnostic sample of the subject indicates a morenegative prognosis for the subject.

Finally, the present invention discloses kits for determining whether asubject has a neurodegenerative disease or is at increased risk fordeveloping a neurodegenerative disease.

Additional objects of the present invention will be apparent in view ofthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence of the saitohin-Q (STH-Q) gene(SEQ ID NO:1). The protein start (atg) and stop (tag) codons areunderlined. The nucleotide sequence of STH-Q differs from that ofsaitohin-R at the 126^(th) nucleotide which, in STH-Q, is A (capitalizedand bolded).

FIG. 2 depicts the nucleotide sequence of the saitohin-R (STH-R) gene(SEQ ID NO:2). The protein start (atg) and stop (tag) codons areunderlined. In STH-R, the 126^(th) nucleotide is G (capitalized andbolded).

FIG. 3 sets forth the predicted amino acid sequence of the STH-Q protein(SEQ ID NO:3). The 126^(th) nucleotide of the STH-Q gene results in a Q(glutamine) at position seven of the STH-Q protein.

FIG. 4 shows the predicted amino acid sequence of the STH-R protein (SEQID NO:4). The difference in the 126^(th) nucleotide of the STH-Q andSTH-R genes changes the Q (glutamine) in the STH-Q protein to an R(arginine) in the STH-R protein.

FIG. 5 sets forth a representative alleleotyping gel of HinFI-digestedPCR products of the genotypes QQ, QR, and RR, according to methodsdescribed below. The polymorphism creates a novel HinF1restrictionenzyme site. HinF1-digested PCR product yields two bands (at 171 bp and55 bp) in subjects with a Q allele, and three bands (at 55 bp, 74 bp,and 97 bp) in individuals with an R allele. Two QQ homozygotes, two QRheterozygotes, and two RR homozygotes are shown.

FIG. 6 illustrates the tau locus and the physical location of thesaitohin gene (vertical bar, above) within the intron downstream of exon9 of the tau gene.

FIG. 7 depicts the open reading frame for the STH protein. The predictedamino acids (three-letter abbreviation) for the nucleotide sequences areshown. At the boxed glutamine (gln) codon [C A A] at amino acid 7, thenucleotide polymorphism (A->G) changes the codon [C G A] to an argininein the STH protein.

FIG. 8 shows the human expression of saitohin and tau in multipletissues and the central nervous system. RT-PCR was performed on theHuman Tissue Rapid-ScanT™ Panel (Origene) (panels A and B) and the HumanBrain Rapid-Scan™ Panel (Origene) (panels C and D) according topublished protocols (11). Saitohin expression is shown in the panels asa single band (panels A and C). In panels B and D, the expression of thetau isoforms is represented by two bands. The upper band consists ofisoforms with exon 10, and the lower band contains the isoforms withoutexon 10. The lanes of multiple tissues in panels A and B are as follows:1—brain; 2—heart; 3—kidney; 4—spleen; 5—liver; 6—colon; 7—lung; 8—smallintestine; 9—muscle; 10—stomach; 11—testis; 12—placenta; 13—salivarygland; 14—thyroid; 15—adrenal; 16—pancreas; 17—ovary; 18—uterus;19—prostate; 20—skin; 21—PBL; 22—bone marrow; 23—fetal brain; and24—fetal liver. The lanes for (panels C and D) are as follows: 1—frontallobe; 2—temporal lobe; 3—cerebellum; 4—hippocampus; 5—substantia nigra;6—caudate nucleus; 7—amygdala; 8—thalamus; 9—hypothalamus; 10—pons;11—medulla; and 12—spinal cord.

FIG. 9 sets forth a Western blot analysis of normal (NC) and Alzheimer'sdisease (AD) subjects with the QQ, QR, and RR genotypes. The top threepanels show immunoblots of whole lysates of IPTG-induced bacteriaexpressing recombinant 6XHis-tagged saitohin (6H-SA), or 6XHis-taggedtau (6H-TAU), or glutathione S-transferase (GST), or GST-saitohin fusionprotein (GST-SA), with antibodies to GST (DT12), 6XHIS (6 HIS), andsaitohin (TS6). The bottom three panels are immunoblots of partiallypurified brain homogenates from QQ, QR, or RR genotypes of AD (QQ-AD,AD-QR, and AD-RR) and normal (QQ-NC, QR-NC) subjects, with left, center,and right panels of STH monoclonal antibodies (11F11, TS6, and 10B3,respectively), according to published protocols (12).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel gene, saitohin (STH), thatis located on chromosome 17, within an intron of the gene encoding themicrotubule-associated protein, tau. As disclosed herein, the saitohingene exists in at least two forms, or alleles, in the human population.In one form, referred to herein as “saitohin-Q (STH-Q)”, the 126^(th)nucleotide is A, which results in a Q (glutamine) at position seven ofthe STH-Q isoform of the STH protein. In the second form of STH,referred to herein as “saitohin-R (STH-R)”, the 126^(th) nucleotide isG, which results in an R (arginine) at position seven of the STH-Risoform of the STH protein.

Accordingly, the present invention provides a saitohin (STH) gene, andan isolated nucleic acid sequence encoding STH protein. The STH gene,and the nucleic acid sequence encoding STH protein, include both theSTH-Q and STH-R alleles. The STH gene may be an “endogenous” STH gene,which is one that originates or arises naturally, from within anorganism. Due to the degeneracy of the genetic code, the STH gene of thepresent invention includes a multitude of nucleic acid substitutionswhich will also encode STH protein, including both the STH-Q and STH-Risoforms. As used herein, an “STH protein” includes, where appropriate,both an STH protein (including both STH-Q and STH-R isoforms) and an“STH analogue”. Unless otherwise indicated, “protein” shall mean aprotein, protein domain, polypeptide, or peptide. An “STH analogue” maybe any protein having functional similarity to the STH protein that is60% or greater (preferably, 70% or greater) in amino-acid-sequencehomology with the STH protein.

The nucleic acid sequence of the present invention may be genomic DNA,cDNA, RNA, antisense DNA, or antisense RNA, and may be derived from anyspecies. The nucleic sequence of the present invention is preferablyderived from a mammalian species, and, more preferably, from a human.

The nucleic acid sequence of the present invention may be the Q alleleof STH, referred to herein as “saitohin-Q (STH-Q)”, in which the126^(th) nucleotide is A, which results in a Q (glutamine) at positionseven of the STH-Q protein. Where the nucleic acid sequence is the STH-Qallele, said nucleic acid sequence preferably comprises the nucleotidesequence of FIG. 1 (including conservative substitutions thereof).“Conservative substitutions”, as used herein, are those amino acidsubstitutions which are functionally equivalent to the substituted aminoacid residue, either because they have similar polarity or stericarrangement, or because they belong to the same class as the substitutedresidue (e.g., hydrophobic, acidic, or basic). The nucleic acid of thepresent invention may encode the STH-Q isoform of the STH protein,comprising the amino acid sequence set forth in FIG. 3.

The present invention further provides an isolated nucleic acid sequencethat hybridizes, preferably under high stringency conditions (e.g.,hybridization to filter-bound DNA in 0.5-M NaHPO₄ at 65° C. and washingin 0.1×SSC/0.1% SDS at 68° C.) or moderate stringency conditions (e.g.,washing in 0.2×SSC/0.1% SDS at 42° C.) (13), to a second nucleic acidthat is complementary to the nucleotide sequence set forth in FIG. 1 ora contiguous fragment thereof. In addition, the present inventionprovides a nucleic acid sequence encoding the STH-Q isoform of the STHprotein having one or more mutations, wherein the mutations result inthe expression of either a non-functional or mutant protein, or in alack of expression altogether. The mutations may be generated by atleast one of the methods selected from the group consisting of pointmutation, insertion mutation, rearrangement, or deletion mutation, or acombination thereof.

The nucleic acid sequence of the present invention may be the R alleleof STH, referred to herein as “saitohin-R (STH-R)”, in which the126^(th) nucleotide is G, which results in an R (arginine) at positionseven of the STH-R isoform of the STH protein. Where the nucleic acidsequence is the STH-R allele, said nucleic acid sequence preferablycomprises the nucleotide sequence of FIG. 2 (including conservativesubstitutions thereof). The nucleic acid of the present invention mayencode the STH-R isoform of the STH protein, comprising the amino acidsequence set forth in FIG. 4.

The present invention further discloses an isolated nucleic acidsequence that hybridizes, preferably under high stringency conditions(e.g., hybridization to filter-bound DNA in 0.5-M NaHPO₄ at 65° C. andwashing in 0.1×SSC/0.1% SDS at 68° C.) or moderate stringency conditions(e.g., washing in 0.2×SSC/0.1% SDS at 42° C.) (2), to a second nucleicacid that is complementary to the nucleotide sequence set forth in FIG.2 or a contiguous fragment thereof. In addition, the present inventionprovides a nucleic acid sequence encoding the STH-R isoform of the STHprotein having one or more mutations, wherein the mutations result inthe expression of either a non-functional or mutant protein, or in alack of expression altogether. The mutations may be generated by atleast one of the methods selected from the group consisting of pointmutation, insertion mutation, rearrangement, or deletion mutation, or acombination thereof.

The present invention also provides an isolated and purified STHprotein. The STH protein may be isolated from tissue (e.g., braintissue) obtained from a subject, or recombinantly produced as describedbelow. The STH protein includes both the STH-Q and STH-R isoformsdisclosed herein.

The protein of the present invention may be the Q isoform of the STHprotein, or saitohin-Q (STH-Q), in which there is a Q (glutamine) atposition seven. Where the protein is the STH-Q isoform, said proteinpreferably comprises the amino acid sequence set forth in FIG. 3.Alternatively, the STH-Q isoform of the STH protein may be encoded bythe nucleotide sequence set forth in FIG. 1. The present invention isfurther directed to a purified protein encoded by a nucleic acidsequence that hybridizes under high stringency or moderate stringencyconditions to a second nucleic acid sequence that is complementary tothe nucleotide sequence set forth in FIG. 1 or a contiguous fragmentthereof.

The protein of the present invention also may be the R isoform of theSTH protein, or saitohin-R (STH-R), in which there is an R (arginine) atposition seven. Where the protein is the STH-R isoform, said proteinpreferably comprises the amino acid sequence set forth in FIG. 4.Alternatively, the STH-Q isoform of the STH protein may be encoded bythe nucleotide sequence set forth in FIG. 2. The present invention isfurther directed to a purified protein encoded by a nucleic acidsequence that hybridizes under high stringency or moderate stringencyconditions to a second nucleic acid sequence that is complementary tothe nucleotide sequence set forth in FIG. 2 or a contiguous fragmentthereof.

Additionally, the present invention provides agents that bind to an STHprotein, including both the STH-Q and STH-R isoforms of said STHprotein. The agent may include, without limitation, an antibody, acompound, a drug, a Fab fragment, a F(ab′)₂ fragment, a molecule, anucleic acid, a protein (including a growth factor), a polypeptide, apeptide, a nucleic acid (including DNA, RNA, mRNA, antisense RNA), andany combinations thereof. Furthermore, the agent that binds to the STHprotein, including both the STH-Q and STH-R isoforms, may be eithernatural or synthetic. A Fab fragment is a univalent antigen-bindingfragment of an antibody, which is produced by papain digestion. AF(ab′)₂ fragment is a divalent antigen-binding fragment of an antibody,which is produced by pepsin digestion. Agents that bind to the STHprotein may be identified or screened by contacting the protein with theagent of interest, and assessing the ability of the agent to bind to theprotein.

The agent of the present invention is preferably an antibody specificfor, or immunoreactive with, STH protein, including the STH-Q and theSTH-R isoforms. The antibody of the present invention may be monoclonalor polyclonal, and may be produced by techniques well known to thoseskilled in the art. The antibody of the present invention may beincorporated into kits which include an appropriate labeling system,buffers, and other necessary reagents for use in a variety of detectionand diagnostic applications. Labeling of the antibody of the presentinvention may be accomplished by standard techniques using one of thevariety of different chemiluminescent and radioactive labels known inthe art.

The present invention further provides a method for producing anantibody specific for the STH protein, including both the STH-Q andSTH-R isoforms, comprising the steps of: (a) immunizing a mammal withSTH protein (e.g., STH-Q or STH-R); and (b) purifying antibody from atissue of the mammal or from a hybridoma made using tissue of themammal. For example, a polyclonal antibody may be produced by immunizinga rabbit, mouse, or rat with purified STH (e.g., STH-Q or STH-R).Thereafter, a monoclonal antibody may be produced by removing the spleenfrom the immunized rabbit, mouse, or rat, and fusing the spleen cellswith myeloma cells to form a hybridoma which, when grown in culture,will produce a monoclonal antibody. Also provided is an antibodyproduced by this method.

The present invention further discloses agents that bind to a nucleicacid encoding STH protein, including both the STH-Q and STH-R alleles.Suitable agents include, but are not limited to, an antibody, acompound, a drug, a Fab fragment, a F(ab′)₂ fragment, a molecule, anucleic acid, a protein, a polypeptide, a peptide, a nucleic acid(including DNA, RNA, mRNA, antisense RNA), and any combinations thereof.The agents that bind to the nucleic acid encoding STH may inhibit orpromote expression of the nucleic acid. Such agents may be discovered bya method for screening for an agent that binds to a nucleic acidencoding STH (e.g., the STH-Q allele or the STH-R allele), comprisingcontacting the nucleic acid with an agent of interest, and assessing theability of the agent to bind to the nucleic acid. An agent that inhibitsor promotes the expression of a nucleic acid encoding STH may bescreened by contacting a host cell transformed with a vector comprisingthe nucleic acid, and assessing the agent's effect on expression of thenucleic acid.

The present invention also provides nucleic acid probes and mixturesthereof that hybridize to nucleic acid encoding STH protein, includingboth the STH-Q and STH-R alleles. Such probes may be prepared by avariety of techniques known to those skilled in the art, including,without limitation, PCR and restriction-enzyme digestion of nucleic acidencoding STH (e.g., the STH-Q allele or the STH-R allele); and automatedsynthesis of oligonucleotides whose sequences correspond to selectedportions of the nucleotide sequence of nucleic acid encoding STH, usingcommercially-available oligonucleotide synthesizers such as the AppliedBiosystems Model 392 DNA/RNA synthesizer.

The nucleic acid probes of the present invention also may be prepared sothat they contain at least one point, insertion, rearrangement, ordeletion mutation, or a combination thereof, to correspond to mutationsof the STH gene.

The nucleic acid probes of the present invention may be DNA or RNA, andmay vary in length from about 8 nucleotides to the entire length of thenucleic acid encoding STH (e.g., the STH-Q allele or the STH-R allele).Preferably, the probes are 8 to 30 nucleotides in length. Labeling ofthe nucleic acid probes may be accomplished using one of a number ofmethods known in the art, including, without limitation, PCR, nicktranslation, end labeling, fill-in end labeling, polynucleotide kinaseexchange reaction, random priming, or SP6 polymerase (for riboprobepreparation), and one of a variety of labels, including, withoutlimitation, radioactive labels such as ³⁵S, ³²P, or ³H andnonradioactive labels such as biotin, fluorescein (FITC), acridine,cholesterol, or carboxy-X-rhodamine (ROX). Combinations of two or morenucleic probes, corresponding to different or overlapping regions ofnucleic acid encoding STH, also may be included in kits for use in avariety of detection and diagnostic applications.

The present invention is further directed to a vector comprising anucleic acid sequence encoding STH protein. The nucleic acid sequenceencoding STH protein may, for example, be the STH-Q allele or the STH-Rallele. Where the nucleic acid sequence of the vector is the STH-Qallele, said nucleic acid sequence may comprise the nucleotide sequenceof FIG. 1 or a contiguous fragment thereof. Alternatively, the nucleicacid sequence of the vector may comprise a nucleic acid sequence thathybridizes under high stringency or moderate stringency conditions to anucleic acid sequence that is complementary to the nucleotide sequenceset forth in FIG. 1, or to a contiguous fragment thereof. Where thenucleic acid sequence of the vector is the STH-R allele, said nucleicacid sequence may comprise the nucleotide sequence of FIG. 2 or acontiguous fragment thereof. Alternatively, the nucleic acid sequence ofthe vector may comprise a nucleic acid sequence that hybridizes underhigh stringency or moderate stringency conditions to a nucleic acidsequence that is complementary to the nucleotide sequence set forth inFIG. 2, or to a contiguous fragment thereof.

The vector of the present invention may be constructed by insertingnucleic acid encoding STH (e.g., the STH-Q or the STH-R allele) into asuitable vector nucleic acid operably linked to an expression controlsequence, as described below. The term “inserted”, as used herein, meansthe ligation of a foreign DNA fragment with vector DNA, by techniquessuch as the annealing of compatible cohesive ends generated byrestriction endonuclease digestion, or by the use of blunt-end ligationtechniques. Other methods of ligating DNA molecules will be apparent toone skilled in the art.

The vector of the present invention may be derived from a number ofdifferent sources, including plasmids, viral-derived nucleic acids,lyric bacteriophage derived from phage lambda, cosmids, or filamentoussingle-stranded bacteriophages such as M13. Depending upon the type ofhost cell into which the vector is introduced, vectors may be bacterialor eukaryotic. Bacterial vectors are derived from many sources,including the genomes of plasmids and phages. Eukaryotic vectors areconstructed from a number of different sources, e.g., yeast plasmids andviruses. Some vectors, referred to as shuttle vectors, are capable ofreplicating in both bacteria and eukaryotes. The nucleic acid from whichthe vector is derived is usually greatly reduced in size, such that onlythose genes essential for its autonomous replication remain. Thisreduction in size enables the vectors to accommodate large segments offoreign DNA. Examples of suitable vectors into which nucleic acidencoding STH (e.g., the STH-Q or the STH-R allele) can be insertedinclude, but are not limited to, pCGS, pBR322, pUC18, pUC19, pHSV-106,pJS97, pJS98, M13 mp18, M13 mp19, pSPORT 1, pGem, pSPORT 2, pSV●SPORT 1,pBluescript II, λZapII, λgt10, λgt11, λgt22A, and λZIPLOX. Othersuitable vectors will be obvious to one skilled in the art.

The vector of the present invention may be introduced into a host cell.Accordingly, the present invention further provides a host celltransformed with the vector of the present invention. The term “hostcell”, as used herein, means the bacterial or eukaryotic cell into whichthe vector is introduced. The term “transform” denotes the introductionof a vector into a bacterial or eukaryotic host cell. Additionally, asused herein, the term “introduction” is a general term indicating thatone of a variety of means has been used to allow the vector to enter theintracellular environment of the host cell in such a way that thenucleic acid exists in stable form, and may be expressed, therein. Assuch, it encompasses transformation of bacterial cells, as well astransfection, transduction, and related methods in eukaryotic cells. Thevector of the present invention may exist in integrated or unintegratedform within the host cell. When in unintegrated form, the vector iscapable of autonomous replication.

Any one of a number of suitable bacterial or eukaryotic host cells maybe transformed with the vector of the present invention. Examples ofsuitable host cells are known to one skilled in the art, and include,without limitation, bacterial cells such as Escherichia coli strainsc600, c600hfl, HB101, LE392, Y1090, JM103, JM109, JM101, JM107, Y1088,Y1089, Y1090, Y1090(ZZ), DM1, PH10B, DH11S, DH125, RR1, TB1 and SURE,Bacillus subtilis, Agrobacterium tumefaciens, Bacillus megaterium; andeukaryotic cells such as Pichia pastoris, Chlamydomonas reinhardtii,Cryptococcus neoformans, Neurospora crassa, Podospora anserina,Saccharomyces cerevisiae, Saccharomyces pombe, Uncinula necator,cultured insect cells, cultured chicken fibroblasts, cultured hamstercells, cultured human cells such as HT1080, MCF7, and 143B, and culturedmouse cells such as EL4 and NIH3T3 cells.

Some bacterial and eukaryotic vectors have been engineered so that theyare capable of expressing inserted nucleic acids to high levels withinthe host cell. An “expression cassette” or “expression controlsequence”, comprising nucleic acid encoding a STH protein (e.g., theSTH-Q or the STH-R allele) operably linked or under the control oftranscriptional and translational regulatory elements (e.g., a promoter,ribosome binding site, operator, or enhancer), can be made and used forexpression of STH protein (e.g., the STH-Q or the STH-R isoform) invitro or in vivo. As used herein, “expression” refers to the ability ofthe vector to transcribe the inserted nucleic acid into mRNA, so thatsynthesis of the protein encoded by the inserted nucleic acid can occur.The choice of regulatory elements employed may vary, depending on suchfactors as the host cell to be transformed and the desired level ofexpression.

For example, in vectors used for the expression of a gene in a bacterialhost cell such as Escherichia coli, the lac operator-promoter or the tacpromoter is often used. Eukaryotic vectors use promoter-enhancersequences of viral genes, especially those of tumor viruses. Severalpromoters for use in mammalian cells are known in the art. Examples ofthese promoters include, without limitation, the phosphoglycerate (PGK)promoter, the simian virus 40 (SV40) early promoter, the Rous sarcomavirus (RSV) promoter, the adenovirus major late promoter (MLP), and thehuman cytomegalovirus (CMV) immediate early 1 promoter. However, anypromoter that facilitates suitable expression levels can be used in thepresent invention. Inducible promoters (e.g., those obtained from theheat shock gene, metallothionine gene, beta-interferon gene, or steroidhormone responsive genes, including, without limitation, the lacoperator-promoter in E. coli and metallothionine or mouse mammary tumorvirus promoters in eukaryotic cells) may be useful for regulatingtranscription based on external stimuli.

Vectors suitable for expression in a host cell of nucleic acid encodingSTH (e.g., the STH-Q or the STH-R allele) are well-known to one skilledin the art, and include pET-3d (Novagen), pProEx-1 (Life Technologies),pFastBac 1 (Life Technologies), pSFV (Life Technologies), pcDNA II(Invitrogen), pSL301 (Invitrogen), pSE280 (Invitrogen), pSE380(Invitrogen), pSE420 (Invitrogen), pTrcHis A,B,C (Invitrogen), pRSETA,B,C (Invitrogen), pYES2 (Invitrogen), pAC360 (Invitrogen), pVL1392 andpVL1392 (Invitrogen), pCDM8 (Invitrogen), pcDNA 1 (Invitrogen), pcDNAI(amp) (Invitrogen), pZeoSV (Invitrogen), pcDNA 3 (Invitrogen), pRc/CMV(Invitrogen), pRc/RSV (Invitrogen), pREP4 (Invitrogen), pREP7(Invitrogen), pREP8 (Invitrogen), pREP9 (Invitrogen), pREP10(Invitrogen), pCEP4 (Invitrogen), pEBVHis (Invitrogen), and λPop6. Othervectors will be apparent to one skilled in the art.

The vector of the present invention may be introduced into a host cellusing conventional procedures known in the art, including, withoutlimitation, electroporation, DEAE Dextran transfection, calciumphosphate rransfection, monocationic liposome fusion, polycationicliposome fusion, protoplast fusion, creation of an in vivo electricalfield, DNA-coated microprojectile bombardment, injection withrecombinant replication-defective viruses, homologous recombination, invivo gene therapy, ex vivo gene therapy, viral vectors, and naked DNAtransfer, or any combination thereof. For the purposes of gene transferinto a host cell, tissue, or subject, a recombinant vector containingnucleic acid encoding STH (e.g., the STH-Q or the STH-R allele) may becombined with a sterile aqueous solution that is preferably isotonicwith the blood of the recipient. Such formulations may be prepared bysuspending the recombinant vector in water containingphysiologically-compatible substances, such as sodium chloride, glycine,and the like, and having buffered pH compatible with physiologicalconditions, to produce an aqueous solution, then rendering the solutionsterile. In a preferred embodiment of the invention, the recombinantvector is combined with a 20-25% sucrose in saline solution inpreparation for introduction into a mammal.

The present invention further provides a method of making recombinantSTH protein, comprising the steps of: (a) introducing into a suitablebacterial or eukaryotic host cell a nucleic acid sequence encoding STH(e.g., the STH-Q or the STH-R allele), or a nucleic acid that hybridizesunder high stringency conditions or moderate stringency conditions to asecond nucleic acid that is complementary to the nucleotide sequence setforth in FIG. 1 or a contiguous fragment thereof, or a nucleic acid thathybridizes under high stringency conditions or moderate stringencyconditions to a second nucleic acid that is complementary to thenucleotide sequence set forth in FIG. 2 or a contiguous fragmentthereof; (b) maintaining the host cell under conditions such that thenucleic acid sequence is expressed to produce STH protein (e.g., theSTH-Q isoform or the STH-R isoform); and (c) recovering the recombinantSTH protein from the culture medium, from the host cells, or from celllysate. As used herein, the term “recombinant” refers to STH protein(e.g., the STH-Q isoform or the STH-R isoform) produced by purificationfrom a host cell transformed with a vector capable of directing itsexpression to a high level. In the method of the present invention, anucleic acid sequence encoding STH (e.g., the STH-Q allele or the STH-Rallele) may be introduced into a suitable host cell by any of theabove-described methods.

A variety of methods of growing host cells transformed with a vector areknown to those skilled in the art. The type of host cell (i.e.,bacterial or eukaryotic) is the primary determinant of both the methodto be utilized and the optimization of specific parameters relating tosuch factors as temperature, trace nutrients, humidity, and growth time.Depending on the vector used, the host cells may have to be induced bythe addition of a specific compound at a certain point in the growthcycle, in order to initiate expression of the nucleic acid contained inthe vector. Examples of compounds used to induce expression of thenucleic acid contained in the vector are known to one skilled in theart, and include, without limitation, IPTG, zinc, and dexamethasone.Using standard methods of protein isolation and purification, such asammonium sulfate precipitation and subsequent dialysis to remove salt,followed by fractionation according to size, charge of the protein atspecific pH values, affinity methods, etc., recombinant STH (e.g., theSTH-Q isoform or the STH-R isoform) may be extracted from suitable hostcells transformed with a vector capable of expressing nucleic acidencoding STH (e.g., the STH-Q allele or the STH-R allele).

It is also within the confines of the present invention to provide atransgenic non-human animal whose genome comprises a disruption in theSTH gene, or a transgenic non-human animal that overexpresses STHprotein (e.g., the STH-Q or STH-R isoform). The STH gene is known toexist in non-human animals, particularly mice. The non-human animal maybe any suitable animal (e.g., cat, cattle, dog, horse, goat, rodent, andsheep), but is preferably a rodent. More preferably, the non-humananimal is a rat or a mouse. The transgenic non-human animal of thepresent invention may be produced by a variety of techniques forgenetically engineering transgenic animals, including those known in theart.

As used herein, the term “transgenic non-human animal” refers to agenetically-engineered non-human animal, produced by experimentalmanipulation, whose genome has been altered by introduction of atransgene. As further used herein, the term “transgene” refers to anucleic acid (e.g., DNA or a gene) that has been introduced into thegenome of an animal by experimental manipulation, wherein the introducedgene is not endogenous to the animal, or is a modified or mutated formof a gene that is endogenous to the animal. The modified or mutated formof an endogenous gene may be produced through human intervention (e.g.,by introduction of a point mutation, introduction of a frameshiftmutation, deletion of a portion or fragment of the endogenous gene,insertion of a selectable marker gene, insertion of a termination codon,etc.). A transgenic non-human animal may be produced by several methodsinvolving human intervention, including, without limitation,introduction of a transgene into an embryonic stem cell, newlyfertilized egg, or early embryo of a non-human animal; integration of atransgene into a chromosome of the somatic and/or germ cells of anon-human animal; and any of the methods described herein.

In one embodiment, the transgenic animal of the present invention has agenome in which the STH gene has been selectively inactivated, resultingin a disruption in its endogenous STH gene. As used herein, a“disruption” refers to a mutation (i.e., a permanent, transmissablechange in genetic material) in the STH gene that prevents normalexpression of functional STH protein (e.g., it results in expression ofa mutant STH protein; it prevents expression of a normal amount of STHprotein; or it prevents expression of STH protein). Examples of adisruption include, without limitation, a point mutation, introductionof a frameshift mutation, deletion of a portion or fragment of theendogenous gene, insertion of a selectable marker gene, and insertion ofa termination codon. As used herein, the term “mutant” is used herein torefer to a gene (or its gene product) which exhibits at least onemodification in its sequence (or its functional properties) as comparedwith the wild-type gene (or its gene product). In contrast, the term“wild-type” refers to the characteristic genotype (or phenotype) for aparticular gene (or its gene product), as found most frequently in itsnatural source (e.g., in a natural population). A wild-type animal, forexample, expresses functional STH.

Selective inactivation in the transgenic non-human animal of the presentinvention may be achieved by a variety of methods, and may result ineither a heterozygous disruption (wherein one STH gene allele (e.g., theSTH-Q allele or the STH-R allele) is disrupted, such that the resultingtransgenic animal is heterozygous for the mutation) or a homozygousdisruption (wherein both STH alleles —STH-Q and STH-R—are disrupted,such that the resulting transgenic animal is homozygous for themutation).

In one embodiment of the present invention, the endogenous STH gene ofthe transgenic animal is disrupted through homologous recombination witha nucleic acid sequence that encodes a region common to STH geneproducts. By way of example, the disruption through homologousrecombination may generate a knockout mutation in the STH gene,particularly a knockout mutation wherein at least one deletion has beenintroduced into at least one exon of the STH gene. Additionally, adisruption in the STH gene may result from insertion of a heterologousselectable marker gene into the endogenous STH gene.

The method for creating a transgenic non-human animal having a knockoutmutation in its STH gene may comprise the following steps: (a)generating an STH targeting vector; (b) introducing the STH targetingvector into a recipient cell of a non-human animal, to produce a treatedrecipient cell; (c) introducing the treated recipient cell into ablastocyst of a non-human animal, to produce a treated blastocyst; (d)introducing the treated blastocyst into a pseudopregnant non-humananimal; (e) allowing the transplanted blastocyst to develop to term; (f)identifying a transgenic non-human animal whose genome comprises aknockout disruption in its endogenous STH gene; and (g) breeding thetransgenic non-human animal to obtain a transgenic non-human animalexhibiting decreased expression of STH protein relative to wild-type.

It is also within the confines of the present invention to provide atransgenic non-human animal that overexpresses STH (e.g., the STH-Qisoform or the STH-R isoform). The non-human animal may be any suitableanimal (e.g., cat, cattle, dog, horse, goat, rodent, and sheep), but ispreferably a rodent. More preferably, the non-human animal is a rat or amouse. The transgenic non-human animal of the present invention may beproduced a variety of techniques for genetically engineering transgenicanimals, including those known in the art. For example, the transgenicanimal may be produced by several methods involving human intervention,including, without limitation, introduction of a transgene into anembryonic stem cell, newly fertilized egg, or early embryo of anon-human animal; integration of a transgene into a chromosome of thesomatic and/or germ cells of a non-human animal; and any of the methodsdescribed herein.

After the transgenic animals of the present invention (i.e., atransgenic non-human animal whose genome comprises a disruption in theSTH gene and a transgenic non-human animal that overexpresses STH-Q orSTH-R) have been produced, each may be analyzed to determine if thetransgene resulted in a pathology (e.g., the accumulation of neuriticplaques or neurofibrillary tangles). If pathologies do not develop inthe animals, the transgenic animals may be crossed with other transgenicanimals that do develop pathologies (e.g., the accumulation of neuriticplaques or neurofibrillary tangles), to determine whether the presenceof the transgene accelerates the pathology in question. For example, theinventors believe that the STH-R isoform of STH protein may beresponsible for accelerating such pathologies as neuritic plaques andneurofibrillary tangles.

The present invention also provides a method for determining whether asubject has or had a neurodegenerative disease (in the case of autopsy),or is at increased risk for developing a neurodegenerative disease. Asused herein, the “subject” is a mammal (either living or deceased),including, without limitation, a cow, dog, human, monkey, mouse, pig, orrat, but is preferably a human. Examples of neurodegenerative diseasesinclude, without limitation, Alzheimer's disease, amyotrophic lateralsclerosis (Lou Gehrig's Disease), Binswanger's disease, corticobasaldegeneration (CBD), dementia lacking distinctive histopathology (DLDH),frontotemporal dementia (FTD), Huntington's chorea, multiple sclerosis,myasthenia gravis, Parkinson's disease, Pick's disease, and progressivesupranuclear palsy (PSP). In the method of the present invention, theneurodegenerative disease is preferably Alzheimer's disease (AD).

As disclosed herein, the STH-R genotype is associated with an increasedrisk of developing Alzheimer's disease. The homozygous STH-R genotype isassociated with the highest probability of developing AD. Therefore,detection of the presence of the STH-R allele in a diagnostic sample ofa subject is indicative that the subject has a neurodegenerative disease(e.g., AD), or had a neurodegenerative disease (e.g., AD) (in the caseof autopsy), or is at increased risk for developing a neurodegenerativedisease (e.g., AD).

Accordingly, the method of the present invention comprises assaying adiagnostic sample of the subject for the presence of one or more allelesof the saitohin (STH) gene, wherein detection of the presence of theSTH-R allele is indicative that the subject has a neurodegenerativedisease (e.g., AD), or had a neurodegenerative disease (e.g., AD) (inthe case of autopsy), or is at increased risk for developing aneurodegenerative disease (e.g., AD). As used herein, the term “atincreased risk for developing a neurodegenerative disease” refers to asubject who is/was predisposed to a neurodegenerative disease, has/had agenetic susceptibility for a neurodegenerative disease, or is/was morelikely to develop a neurodegenerative disease than a subject in whom theSTH-R allele of the STH gene is not present (i.e., a subject homozygousfor STH-Q). In a preferred embodiment of the present invention, themethod described herein is used to determine whether a subject has AD orhad AD (in the case of autopsy), or is at increased risk for developingAD, and detection of the presence of the STH-R allele is indicative thatthe subject has AD (or had AD) or is at increased risk for developingAD. In one embodiment, the AD is late-onset AD (LOAD).

Screening for the presence of STH-R in a diagnostic sample of a subjectoffers a non-invasive method for determining whether a subject has AD orhad AD (in the case of autopsy), or is at an increased risk ofdeveloping AD. It is contemplated that the method of the presentinvention may be used alone, or in addition to other screens for AD riskfactors (e.g., ApoE4) (see, e.g., U.S. Pat. Nos. 5,716,828; 5,767,248;and 6,136,530).

According to the method of the present invention, the diagnostic sampleof a subject may be assayed in vitro or in vivo. In accordance with thepresent invention, where the assay is performed in vitro, a diagnosticsample from the subject may be removed using standard procedures. Thediagnostic sample may be tissue, particularly any brain tissue, kidneytissue, muscle tissue, nervous tissue, retinal tissue, or soft tissue,which may be removed by standard biopsy. In addition, the diagnosticsample may be a bodily fluid, including blood, cerebrospinal fluid,pericardial fluid, peritoneal fluid, saliva, serum, and urine. In apreferred embodiment, the diagnostic sample is blood. The diagnosticsample may be taken, for example, from a subject or patient suspected ofhaving a neurodegenerative disease (e.g., AD), a subject or patient notknown to have a neurodegenerative disease (e.g., AD), a subject orpatient whose family has a history of neurodegenerative disease (e.g.,AD), a subject or patient who exhibits cognitive decline, and elderlysubjects or patients.

In the method of the present invention, the diagnostic sample of asubject may be assayed for the presence of one or more alleles of theSTH gene (e.g., the STH-Q allele or the STH-R allele) using assays anddetection methods readily determined from the known art, including,without limitation, immunological techniques, hybridization analysis ofnucleic acid (e.g., genomic DNA) extracted from the diagnostic sampletaken from the subject, fluorescence imaging techniques, radiationdetection, polymerase chain reaction (PCR), ligase chain reaction (LCR),RIA assay, and ELISA assay.

Nucleic acid may be isolated from a diagnostic sample of a subject usingstandard techniques known to one of skill in the art. Isolated nucleicacid may be amplified by procedures known in the art, including, withoutlimitation, ligase chain reaction (LCR) (21) and polymerase chainreaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188). For example, in the method of the presentinvention, genomic DNA encoding part or all of the STH gene may beisolated from a diagnostic sample of a subject, and amplified using atleast one pair of STH-specific or STH-region-specific oligonucleotideprimers, such as those disclosed herein. The amplified DNA that isthereby generated then may be incubated with a restriction enzyme,including any disclosed herein, that is capable of cleaving DNA at sitesspecific to the STH region to form a digest. Thereafter, the size of theDNA fragments in the digest may be determined. The presence of a DNAfragment having a size that differs from the size of a DNA fragment in acontrol sample lacking the STH-R allele indicates the presence of theSTH-R allele in the diagnostic sample of the subject.

According to this method of the present invention, the hybridizationanalysis may be conducted using Northern blot analysis of mRNA.Additionally, this method of the present invention may be conducted byperforming a Southern blot analysis of DNA using one or more nucleicacid probes that hybridize to nucleic acid encoding STH (e.g., the STH-Qallele or the STH-R allele). The nucleic acid probes may be prepared bya variety of techniques known to those skilled in the art, including,without limitation, the following: restriction enzyme digestion ofnucleic acid encoding STH protein; automated synthesis ofoligonucleotides having sequences which correspond to selected portionsof the nucleotide sequence of the STH gene, using commercially-availableoligonucleotide synthesizers, such as the Applied Biosystems Model 392DNA/RNA synthesizer; and any methods disclosed herein.

The STH nucleic acid used in the probes may be derived from mammalian,preferably human, STH. The nucleic acid probes used in the method of thepresent invention may be DNA or RNA, and may vary in length from about 8nucleotides to the entire length of the STH nucleic acid. In addition,the probes may be prepared in accordance with probe preparation methodsdescribed above. Furthermore, the nucleic acid probes of the presentinvention may be labeled with one or more detectable markers. Labelingof the nucleic acid probes may be accomplished using one of a number ofmethods known in the art, including those described above, along withone of a variety of labels, including those described above.Combinations of two or more nucleic acid probes (or primers),corresponding to different or overlapping regions of the STH nucleicacid, also may be used to assay a diagnostic sample for STH expression,using, for example, PCR or RT-PCR.

As disclosed herein, the presence of the STH-R isoform of the STHappears to increase the risk of developing AD. Thus, genotyping is alsopossible by direct examination of the STH protein present in either thetissue or bodily fluid of a subject. Accordingly, in the method of thepresent invention, the diagnostic sample of a subject also may beassayed for the presence of one or more alleles of the STH gene byassaying for expression of one or more isoforms of the STH protein(e.g., the STH-Q isoform and the STH-R isoform). As used herein,“expression” means the transcription of the STH gene into at least onemRNA transcript, or the translation of at least one mRNA into an STHprotein, as defined above. Accordingly, a diagnostic sample may beassayed for STH expression by assaying for STH protein (as definedabove), STH cDNA, or STH mRNA. The appropriate form of STH will beapparent based on the particular techniques discussed herein.

Protein may be isolated and purified from the diagnostic sample of thepresent invention using standard methods known in the art, including,without limitation, extraction from a tissue (e.g., with a detergentthat solubilizes the protein) where necessary, followed by affinitypurification on a column, chromatography (e.g., FTLC and HPLC),immunoprecipitation (with an antibody specific to STH), andprecipitation (e.g., with isopropanol and a reagent such as Trizol).Isolation and purification of the protein may be followed byelectrophoresis (e.g., on an SDS-polyacrylamide gel).

In accordance with the method of the present invention, a diagnosticsample of a subject may be assayed for STH expression, and STHexpression may be detected in a diagnostic sample, using assays anddetection methods readily determined from the known art, including,without limitation, immunological techniques, hybridization analysis,fluorescence imaging techniques, radiation detection, PCR, LCR, RIAassay, and ELISA assay. For example, according to the method of thepresent invention, a diagnostic sample of the subject may be assayed forSTH expression using an agent reactive with STH. As used herein,“reactive” means the agent has affinity for, binds to, or is directedagainst STH. As further used herein, an “agent” shall include a protein,polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fabfragment, F(ab′)₂ fragment, molecule, compound, antibiotic, drug, any ofthe agents disclosed above, and any combinations thereof. Preferably,the agent of the present invention is labeled with a detectable marker.

In one embodiment of the present invention, the agent reactive with STHis an allele-specific antibody (e.g., specific for the STH-Q allele orspecific for the STH-R allele). As used herein, the antibody of thepresent invention may be polyclonal or monoclonal. In addition, theantibody of the present invention may be produced by techniques wellknown to those skilled in the art, including any of those describedabove. The antibodies used herein may be labeled with a detectablemarker. Labeling of an antibody may be accomplished using one of avariety of labeling techniques, including those known in the art andthose described above. The detectable marker of the present inventionmay be any of those known in the art, as well as any above-describeddetectable markers. Preferably, the agent of the present invention is ahigh-affinity antibody labeled with a detectable marker.

Where the agent of the present invention is an antibody reactive withSTH, a diagnostic sample taken from the subject may be purified bypassage through an affinity column which contains STH antibody as aligand attached to a solid support such as an insoluble organic polymerin the form of a bead, gel, or plate. The antibody attached to the solidsupport may be used in the form of a column. Examples of suitable solidsupports include, without limitation, agarose, cellulose, dextran,polyacrylamide, polystyrene, sepharose, or other insoluble organicpolymers. The STH antibody may be further attached to the solid supportthrough a spacer molecule, if desired. Appropriate binding conditions(e.g., temperature, pH, and salt concentration) may be readilydetermined by the skilled artisan. In one embodiment, the STH antibodyis attached to a sepharose column, such as Sepharose 4B.

Where the agent is an antibody, a diagnostic sample of the subject maybe assayed for STH expression using binding studies that utilize one ormore antibodies immunoreactive with STH, along with standardimmunological detection techniques. For example, the STH protein elutedfrom the affinity column may be subjected to an ELISA assay, an RIAassay, Western blot analysis, flow cytometry, or any otherimmunostaining method employing an antigen-antibody interaction. In apreferred embodiment of the present invention, the diagnostic sample isassayed for STH expression using an ELISA assay.

In accordance with the method of the present invention, the detection ofthe presence of one or more alleles of STH in a diagnostic sample of asubject may be followed by an assay to measure or quantify the relativeand total amounts of STH-Q and STH-R present in the diagnostic sample ofthe subject. Such assays are well known to one of skill in the art, andmay include immunohistochemistry/immunocytochemistry, flow cytometry,mass spectroscopy, Western blot analysis, or an ELISA for measuringamounts of STH protein. For example, to use an immunohistochemistryassay, histological (paraffin-embedded) sections of tissue may be placedon slides, and then incubated with an antibody against STH. The slidesthen may be incubated with a second antibody (against the primaryantibody), which is tagged to a dye or other calorimetric system (e.g.,a fluorochrome, a radioactive agent, or an agent having highelectron-scanning capacity), to permit visualization of STH present inthe sections.

It is contemplated that the diagnostic sample in the present inventionfrequently will be assayed for STH expression not by the subject orpatient, nor by his/her consulting physician, but by a laboratorytechnician or other clinician. Accordingly, the method of the presentinvention further comprises providing to a subject's or patient'sconsulting physician a report of the results obtained upon assaying adiagnostic sample of the subject or patient for the presence of one ormore alleles of STH.

As disclosed herein, two known alleles of the STH gene (STH-Q and STH-R)are present in the human population. Therefore, there are three possiblegenotypes: (1) homozygous for STH-Q, or QQ; (2) homozygous for STH-R, orRR; and (3) heterozygous, having one copy of STH-Q and one copy ofSTH-R, abbreviated QR. The inventors have found that a correlationexists between the presence of the STH-R genotype in a subject, and thesubject's risk of developing Alzheimer's disease. In particular, thepresence of a single STH-R allele (i.e., the subject is heterozygous forSTH-R, or QR) is associated with an increased risk of developing AD,while the presence of two STH-R alleles (i.e., the subject is homozygousfor STH-R, or RR) is associated with the highest risk of developing AD.

In view of the foregoing, it is also contemplated in the presentinvention that assaying a diagnostic sample of a subject for thepresence of one or more alleles of STH may be a useful means ofproviding information concerning the prognosis of a subject or patientwho has, or may develop, a neurodegenerative disease (e.g., AD).Accordingly, the present invention further provides a method forassessing the prognosis of a subject who has a neurodegenerativedisease, or who may develop a neurodegenerative disease, comprisingassaying a diagnostic sample of the subject for the presence of one ormore alleles of STH, wherein the presence of two STH-R alleles in thediagnostic sample of the subject indicates a more negative prognosis forthe subject.

In accordance with the method of the present invention, theneurodegenerative disease may be any of those described above, includingAlzheimer's disease (AD). In a preferred embodiment, theneurodegenerative disease is AD. In one such preferred embodiment, theAD is late-onset AD (LOAD). The diagnostic sample of the subject may bea tissue or a bodily fluid, as described above, and may be removed fromthe subject by known procedures, including those discussed above. In oneembodiment, the diagnostic sample is a blood sample. Additionally, thediagnostic sample may be assayed either in vitro or in vivo, using allof the various assays, detection methods, and quantification methodsdescribed above. Furthermore, the diagnostic sample of a subject orpatient may be assayed, and the presence of one or more alleles of STHmay be determined, at any time before, during, or following thedetermination that the subject or patient has a neurodegenerativedisease (e.g., AD), or is at increased risk of developing aneurodegenerative disease (e.g., AD).

It is contemplated that the diagnostic sample of the present inventionfrequently will be assayed for the presence of one or more alleles ofSTH not by the subject or patient, nor by his/her consulting physician,but by a laboratory technician or other clinician. Accordingly, themethod of the present invention further comprises providing to asubject's or patient's consulting physician a report of the resultsobtained upon assaying a diagnostic sample of the subject or patient forthe presence of one or more alleles of STH.

The inventors' biochemical results have shown that the STH-R isoform ofthe STH protein accumulates in the brain to a greater extent than doesthe STH-Q isoform. Therefore, detection of increased levels of STHappears to be indicative of the presence of the STH-R genotype. Sincethe STH-R isoform of the STH protein accumulates in the brain andpossibly other tissues, it is reasonable to expect that thisaccumulation will be reflected by increased levels in the cerebrospinalfluid and blood. Accordingly, detection of increased risk of developmentof AD may simply require only the measurement of levels of STH proteinin a diagnostic sample of a subject. Elevated levels of STH protein thenwould be indicative of the presence of at least one copy of the STH-Rallele sequence in the subject.

In view of the foregoing, it is also within the confines of the presentinvention to provide a method for determining whether a subject hasAlzheimer's disease (AD) or had AD (in the case of autopsy), or is atincreased risk for developing AD, comprising assaying a diagnosticsample of the subject for expression of STH protein, wherein detectionof STH expression elevated above normal is indicative that the subjecthas AD (or had AD, in the case of autopsy), or is at increased risk fordeveloping AD.

As used herein, “STH expression elevated above normal” means expressionof STH at a level that is significantly greater than the level expectedfor the same type of diagnostic sample taken from a nondiseased subjector patient (i.e., one who does not have a neurodegenerative disease(e.g., AD) or a detectable increased risk of developing aneurodegenerative disease (e.g., AD)) of the same gender and of similarage. As further used herein, “significantly greater” means that thedifference between the level of STH expression that is elevated abovenormal, and the expected (normal) level of STH, is of statisticalsignificance. Preferably, STH expression elevated above normal isexpression of STH at a level that is at least 10% greater than the levelof STH expression otherwise expected. Where STH expression is expectedto be absent from a particular diagnostic sample taken from a particularsubject or patient, the normal level of STH expression for that subjector patient is nil. Where a particular diagnostic sample taken from aparticular subject or patient is expected to have a low level ofconstitutive STH expression, that low level is the normal level of STHexpression for that subject or patient.

Expected or normal levels of STH expression for a particular diagnosticsample taken from a subject or patient may be easily determined byassaying nondiseased subjects of a similar age and of the same gender.Once the appropriate samples have been obtained, the normal quantitiesof STH expression in men and women may be determined using a standardassay for quantification, such as flow cytometry, Western blot analysis,or an ELISA for measuring protein quantities, as described below. Forexample, an ELISA may be run on each sample in duplicate, and the meansand standard deviations of the quantity of the STH protein may bedetermined. If necessary, additional subjects may be recruited beforethe normal quantities of STH expression are quantified.

In accordance with the method of the present invention, theneurodegenerative disease may be any of those described above, includingAlzheimer's disease (AD). In a preferred embodiment, theneurodegenerative disease is AD. In one such preferred embodiment, theAD is late-onset AD (LOAD). The diagnostic sample of the subject may bea tissue or a bodily fluid, as described above, and may be removed fromthe subject by known procedures, including those discussed above. In oneembodiment, the diagnostic sample is a blood sample. Additionally, thediagnostic sample may be assayed either in vitro or in vivo, using allof the various protein assays, protein detection methods, and proteinquantification methods described above (e.g., by ELISA and RIA). In oneembodiment, the diagnostic sample is assayed using an agent reactivewith STH, as defined above. The agent may be labeled with a detectablemarker, as described above. In a preferred embodiment, the diagnosticsample is assayed using an antibody that selectively binds STH.Preferably, the antibody is labeled with a detectable marker.

Since increased levels of STH protein appear to be associated with anincreased risk of Alzheimer's disease, overexpression of this protein ineither cells in culture or in rodents (by transgenic means) will providevaluable insights into mechanisms by which overexpression of STH proteinincreases risk for Alzheimer's disease. Mice or cell cultures expressingthe STH-R isoform of STH (including the above-described transgenicnonhuman animals and transformed host cells) will be especially valuablein this regard. The STH-R homozygotes in the human population havedemonstrated that the RR genotype is sufficient to cause Alzheimer'sdisease, even in the absence of other risk factors for Alzheimer's, suchas an ApoE4 allele. Thus, mice overexpressing the STH-R isoform of STHcan be expected to develop an Alzheimer's-disease—like condition. Suchmice will be extremely valuable in the development and testing oftherapies for this disease.

The discovery that there is a correlation between the presence of theSTH-R genotype in a subject, and the subject's risk of developingAlzheimer's disease, provides a means of identifying patients who haveAD or another neurodegenerative disease, or who are at increased risk ofdeveloping AD or another neurodegenerative disease, and presents thepotential for commercial application in the form of a test to screen forsusceptibility to the development of a neurodegenerative disease, suchas AD. The development of such a test could provide general screeningprocedures that may assist in the early detection and diagnosis ofneurodegenerative diseases, such as AD, and/or provide a method for thefollow-up of patients who have been diagnosed with a neurodegenerativedisease (e.g., AD) or identified as being at increased risk ofdeveloping a neurodegenerative disease (e.g., AD).

Accordingly, the present invention further provides a kit for use as ascreening assay to identify a subject who has a neurodegenerativedisease (e.g., AD), or who is at increased risk of developing aneurodegenerative disease (e.g., AD). The kit of the present inventioncomprises at least one reagent for use in an assay to detect directlythe presence of a nucleic acid sequence encoding one or more alleles ofSTH, or at least one reagent for use in an assay to detect the presenceof one or more isoforms of STH protein. The kit also comprisesinstructions for using the kit to determine whether the subject hasAlzheimer's disease (AD) or had AD (in the case of autopsy), or is atincreased risk for developing AD.

Such instructions, for example, may provide that the kit may be used toassay a diagnostic sample of a subject for the presence of one or morealleles of saitohin (STH), wherein detection of the presence of theSTH-R allele is indicative that the subject has AD (or had AD, in thecase of autopsy), or is at increased risk for developing AD.Additionally, such instructions also may provide that the kit may beused to assay a diagnostic sample of a subject for expression ofsaitohin (STH) protein, wherein detection of STH expression elevatedabove normal is indicative that the subject has AD (or had AD), or is atincreased risk for developing AD. In one embodiment of the presentinvention, the kit further comprises a container in which the reagentand the instructions are packaged.

A kit designed to detect the presence of a nucleic acid sequenceencoding one or more alleles of STH may contain an agent specificallyreactive with STH. The agent may be any of those described above,including oligonucleotide probes that selectively bind to a nucleic acidsequence encoding the STH-Q allele or the STH-R allele of the STH gene,and may be used in any of the above-described assays or methods fordetecting or quantifying the presence of one or more alleles of STH. Thekit of the present invention also may include allele-specific probesthat can hybridize to amplified fragments of a nucleic acid sequencecorresponding to part or all of the STH gene, and that can be used toidentify the presence of one or more alleles of STH. Furthermore, a kitdesigned to detect the presence of a nucleic acid sequence encoding oneor more alleles of STH may contain primers that hybridize to a nucleicacid sequence corresponding to part or all of the STH gene, and thatpermit the amplification of said sequence (e.g., by LCR, PCR, and otheramplification procedures known in the art).

A kit designed to detect the presence of one or more isoforms of the STHprotein may contain an agent specifically reactive with STH. The agentmay be any of those described above, including an allele-specificantibody that selectively binds the STH-Q or the STH-R isoform of theSTH protein, and may be used in any of the above-described assays ormethods for detecting or quantifying the presence of one or more allelesof STH. The kit of the present invention also may include at least oneallele-specific antibody directed to STH, preferably labeled with adetectable marker, along with a solid support capable of binding STHprotein.

The present invention is described in the following Experimental Detailssection, which is set forth to aid in the understanding of theinvention, and should not be construed to limit in any way the scope ofthe invention as defined in the claims which follow thereafter.

Experimental Details

1. Introduction

In February, the Human Genome Project completed the sequencing for thegenomic clones overlapping the tau locus on Chromosome 17q21, and anexpressed sequence tag (EST) (accession #AA325304) was identified in atau intron approximately 2.5 kb downstream of exon 9 of the tau genethat is shown in FIG. 6. This location was of particular interest to theinventors, because most of the mutations in FTD are in or around thisintron and adjacent exons of tau (3). The EST was found to be expressedin the Marathon cerebellar cDNA library (Clontech), and in a humanoligodendroglioma cell line (that expresses tau). However, mRRNlAisolated from COS7 cells failed to yield an RT-PCR product, therebysuggesting that the mRNA was not expressed in COS7 cells (data notshown).

Using RNA from a human oligodendroglioma cell line, the inventorsdiscovered a new human gene, which exists in at least two forms in thehuman population. This gene, which they have named saitohin (STH), is onchromosome 17, within an intron of the gene encoding themicrotubule-associated protein tau. The protein encoded by the saitohingene is not similar to the microtubule-associated protein tau, nor is itsubstantially similar to any other known protein. Furthermore, searchesof the protein data base have not revealed another protein withsignificant homology to saitohin.

2. Materials and Methods

A. Cloning of Saitohin (STH) Gene

Using RNA from a human oligodendroglioma cell line, the 5′ and 3′ endsof the putative gene were cloned with the Gene Racer kit (Invitrogen).The sequence of the full-length clones revealed a new gene, saitohin(STH) {named in honor of late Dr. Tsunao Saitoh and his lab], which wasintron-less and in the sense direction relative to tau.

B. Analysis of STH Gene Expression

To further characterize STH, the inventors analyzed the gene expressionin 24 human tissues, using the Human Tissue Rapid-Scana Panel (Origene)(FIG. 8). Since STH and tau share a common genetic locus, the inventorshypothesized that STH and tau could be coordinately expressed.Therefore, the expression of tau was also examined. To determine whetherthe location of STH upstream of the alternatively spliced exon 10 wouldaffect its expression, the tau isoforms with exon 10 (FIG. 8, panel A,upper band) and without exon 10 (FIG. 8, panel A, lower band) were alsoexamined. Because tau and STH have a significant overlap in generaltissue expression, an expanded study was performed using the Human BrainRapid-Scana Panel (Origene) to determine the central nervous system(CNS) expression patterns, and to ascertain whether STH and tau arecoordinately expressed in the brain.

In these studies, cDNA was amplified using the “Touchdown” PCR method(11). The primers used were F Cel I (5′-ccc tgt aaa ctc tga cca cac-3′)and R Cel I (5′-cat ggg aag tag ctt ccc tgt-3′). PCR was performed in a50-μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl,1.5 mM MgCl₂, 0.2 μM of each primer, 0.2 mM dNTPs, 2 μl of genomic DNA,and 1 U of Taq polymerase (Invitrogen).

The touchdown PCR method for saitohin consists of the following steps:An incubation at 94° C. for 3 min is followed by a step of 94° C. for 30sec, a step of 65° C. for 30 sec for the initial annealing cycle (ineach subsequent cycle, the annealing temperature is decreased by 0.2°C.), and a step of 72° C. for 30 sec for polymerization. This sequenceis then repeated for 25 cycles. Finally, an additional set of 10 cyclesis performed, consisting of 94° C. for 30 sec, 60° C. for 30 sec, and72° C. for 30 sec. The last cycle is followed by an incubation at 72° C.for 30 sec. For the amplification of tau transcripts, the PCR cocktailand touchdown program were the same as above, except the tau senseprimer was a 21-mer 5′-gcc aac gcc acc agg att cca gca aaa-3′, theantisense primer was a 21-mer 5′-ttt act tcc acc tgg cca cct-3′, and alonger PCR polymerization time of 55 sec was used. Both saitohin and tauPCR products were run on a 2.0% agarose gel with ethidium bromide.

C. PCR Amplification and Restriction-Enzyme Digestion of STH DNA

For the alleleotyping gel (FIG. 5), HinFI-digested PCR products of thegenotypes QQ, QR, and RR were prepared in accordance with knownprotocols. In particular, the genomic DNA of the subjects was extractedfrom frozen brain tissue using a genomic-DNA extraction kit, accordingto manufacturer protocols (Qiagen). The saitohin DNA sequence then wasamplified from genomic DNA from AD and normal control subjects using theabove PCR protocol for saitohin, using the primers F-Cel I and R-Cel I.Sequencing of the PCR products identified a nucleotide polymorphismA->G, changing the amino acid at position 7 from a glutamine (Q) to anarginine (R), thereby creating a new HinFI restriction enzyme site. Forgenotyping, the PCR products were digested with 5 units of HinFI{5′-GANTC-3′} restriction enzyme (New England Biolabs), for 3 h at 37°C., then run on a 2.0% agarose gel with ethidium bromide.

D. STH Monoclonal Antibodies

Saitohin monoclonal antibodies 11F11 (IgG2B), TS6 (IgM), and 10B3 (IgM)were generated as previously described (12) by immunizing mice with bothrecombinant and synthetic peptides of STH. Brain protein samples ofnormal (NC) and Alzheimer's disease (AD) subjects with the QQ, QR, andRR genotypes were homogenized in 1XBS (2 mM PMSF), and protein waspartially purified by size fractionation. Subsequently, the preparedsamples were incubated in a urea sample buffer for 30 min at 37° C., andthen were run on a 15% SDS-PAGE gel (FIG. 9). ECL-Western blot analysiswas performed using previously described protocols (12).

3. Results and Discussion

Nucleotide and amino acid sequence analyses of the STH gene (FIG. 7)revealed that STH encodes a 128-amino-acid protein that appears to haveno clear homology to genes, proteins, signal sequences, or motifs (14).However, the location of STH within the tau locus could provide insightinto its function. Since both STH and tau share a similar expressionpattern, tau and STH could function together as in the example of thecholine acetyltransferase\ vesicular acetylcholine transporter genelocus (15). The mouse genome contains a sequence which is 100% identicalto the saitohin open reading frame, strongly suggesting that this geneis conserved between rodents and humans. The mouse genome contains asequence that is 100% identical to the STH open reading frame, stronglysuggesting that this gene is conserved between rodents and humans.

To further characterize STH, the inventors analyzed gene expression in24 human tissues, using the Human Tissue Rapid-Scan^(a) Panel (Origene).The expression of STH was highest in placenta, muscle, fetal brain andadult brain, with low expression in heart, kidney, stomach, testis, andadrenal gland (FIG. 8, panel A). Since STH and tau share a commongenetic locus, they could be coordinately expressed; therefore, theexpression of tau was also determined. To determine whether the locationof STH upstream of the alternatively spliced exon 10 would affect itsexpression, the tau isoforms with exon 10 and without exon 10 were alsoexamined. As shown in FIG. 8, panel B, the expression of tau was foundto be highest in heart, kidney, muscle, testis, salivary glands, adrenalglands, adult and fetal brain with low expression in placenta, thyroid,prostate, and skin. This is in agreement with other reports of tauexpression (16).

Because tau and STH have a significant overlap in general tissueexpression, an expanded study was performed using the Human BrainRapid-Scan^(a) Panel (Origene) to determine the central nervous system(CNS) expression patterns, and to ascertain whether STH and tau arecoordinately expressed in the brain. FIG. 8, panel C, shows that STH isexpressed in the CNS, with higher expression in the temporal lobe,hypothalamus, medulla, and spinal cord, and with lower expression in theother brain regions. As depicted in FIG. 8, panel D, the expression oftau is high in most of the CNS samples, except in the pons, where theexpression is lower.

Based upon the general tissue and CNS tissue expressions analyzed by theinventors, it appears that tau shares some tissue expression with STH;however, there are some differences. Taken together, the resultsindicate that STH is not under the regulation of the tau promoter, butcould share some regional regulatory elements with the tau gene, whichcould have implications for the function of STH. The location andexpression of STH upstream of the exon 10 does not appear to correlatewith the splicing of exon 10 of tau in the CNS or the other tissues.

The location of STH warrants investigation into its possible role inneurodegenerative disorders, since there is genetic evidence implicatingthe tau locus in many of these diseases. During the sequencing of theSTH gene from human subjects, a nucleotide polymorphism (A->G) wasidentified that changes a glutamine (Q) to arginine (R) at amino acidposition 7 (Q7R) of STH, as shown in FIG. 7. The “G” polymorphism (Rallele) creates a novel HinfI restriction enzyme site that generates adistinctive fragment pattern as compared to the “A” nucleotide (Qallele). A representative gel is shown in FIG. 5.

The distribution of AD and normal control subjects with different STHalleles and frequencies were tabulated in Table 1. The two groups ofsubjects were age-matched, and were limited to Caucasian subjects. TheRR genotype was found at a significantly higher frequency in the ADgroup (16%), as compared to the normal control subjects (0%) (p=0.0232;odds ratio 11.920, by Fisher's Exact Test). The R allele does occur innormal subjects at a frequency of 13%, but at a significantly lowerpercentage compared to AD subjects in which the R allele is 32% (p0.0085; odds ratio 3.109, by Fisher's Exact Test). The average age ofonset for the RR subjects is 83.1 with a range of 77-93, which providesevidence that RR genotype is a risk factor for late onset Alzheimer'sdisease. TABLE 1 STH polymorphism in AD and normal subjects. AD NormalSaitohin Genotype n = 1 n = 30 QQ 26 (51%) 22 (73%) QR 17 (33%)  8 (27%)RR  8 (16%)  0 (0%) Saitohin Alleles n = 102 n = 60 Q 69 (65.6%) 52(86.7%) R 33 (32.4%)  8 (13.3%)A total of 81 subjects were used for the case-control study. The ADgroup (n = 51) and the normal control group (n = 30) of subjects wereautopsy-confirmed and age-matched. The Fisher's Exact Test was used forthe comparisons of the allele and genotype frequencies for the groupsincluded.Normal (average age = 78.83; range 55-97);Alzheimer's disease (AD) (average age = 80.51; range 56-98)

In addition to the STH genotype, the ApoE genotype of the subjects wasalso determined, because allele 4 of the ApoE gene is an important riskfactor for AD, and because the inventors wanted to investigate whetherthe ApoE allele 4 could synergize with the STH genotype (17). The ApoEgenotypes were in close agreement with the frequencies in the generalpopulation, thereby providing evidence against sampling bias (data notshown) (18). In agreement with previous reports, the ApoE4 allele wasfound to be overrepresented in the inventors' AD group, as compared tonormal control subjects shown in Table 2; however, the ApoE alleles wereevenly distributed among the RR, QR, and QQ subjects of the AD group inTable 3, suggesting that there is no association between the ApoE andSTH genotypes (18). TABLE 2 ApoE genetics in AD and normal controls. ADNormal ApoE Genotype n = 1 n = 30 2/2  0 (0%)  0 (0%) 2/3  3 (5.9%)  7(23.3%) 2/4  0 (0%)  0 (0%) 3/3 22 (43.1%) 19 (63.4%) 3/4 20 (39.2%)  4(13.3%) 4/4  6 (11.8%)  0 (0%) ApoE Alleles n = 102 n = 60 2  3 (2.9%) 7 (11.7%) 3 67 (65.7%) 49 (81.7%) 4 32 (31.4%)  4 (6.6%)ApoE genotyping was carried out as previously described (20). TheFisher's Exact Test was used for comparisons of the ApoE4 carrier (p =0.0008; OR = 6.760; CI = 2.062-22.166) and ApoE4 allele frequencies (p =0.0002; OR = 6.4; CI = 2.136-19.178) for the groups included.

TABLE 3 STH genotype compared with ApoE genotype. AD ApoE4 ApoE4 n = 51Negative Positive STH Genotype n = 25 n = 26 QQ 13  13  QR 8 9 RR 4 4Normal ApoE4 ApoE4 n = 30 Negative Positive STH Genotype n = 26 n = 4 QQ20  3 QR 6 1 RR 0 0A logistic regression analysis was performed with the statisticalprogram, SPSS, for the determination of associations of ApoE4 with RRgenotype in the AD and normal populations.

In addition to the AD group, a small number of subjects with otherneurodegenerative disorders, most of which have tau pathology, were alsoexamined for the polymorphism. These results, which are set forth inTable 4, show some interesting trends, and suggest that the STH-Q/Rpolymorphism may not be AD specific. For example, one subject withdementia lacking distinctive histopathology had the RR genotype, therebysuggesting that this genotype is not AD specific. Furthermore, itappears that PSP subjects have an overrepresentation of the QQ genotype,while FTD and Pick's disease have a higher percentage of QR genotype,when compared with normal controls. Further investigation of thesetrends with larger groups of subjects is required to determine theirsignificance. TABLE 4 Saitohin polymorphism in non-AD neurodegenerativedisorders. Saitohin DLDH PSP FTD CBD PICKS ALS Genotype n = 7 n = 6 n =4 n = 4 n = 9 n = 1 QQ 4 (57%) 5 (83%) 1 (25%) 1 (100%) 3 (33%) 1 (100%)QR 2 (29%) 1 (17%) 3 (75%) 0 6 (67%) 0 (0%) RR 1 (14%) 0 (0%) 0 (0%) 0(0%) 0 (0%) 0 (0%)The numbers of subjects were too small for statistical analysis.Subjects with dementia lacking distinctive histopathology (DLDH),frontotemporal dementia (FTD), Pick's disease (PICKS), progressivesupranuclear palsy (PSP), corticobasal degeneration (CBD), andamyotropic lateral sclerosis (ALS), were studied.

The experiments demonstrating STH expression thus far rely upon the useof PCR, which is so sensitive that it is difficult to be sure that bandsresult from legitimate mRNA transcripts or come from a tracecontamination of DNA. When working with an intron-less gene, where thesequence of the mRNA matches up exactly with the genomic DNA encodingthe gene, DNA contamination remains a concern. The consistent failure tofind evidence of STH mRNA in COS7 cells and in several tissues of theMultiple Tissue cDNA panel argues against contamination.

The inventors generated monoclonal antibodies to the predicted protein,produced as either a 6-His tagged recombinant protein, or as aglutathione S-transferase fusion protein in bacteria, in order todemonstrate the existence of a STH protein product. Using a combinationof synthetic peptides from the STH sequence and a deletion mutantprotein, the epitopes recognized by these monoclonal antibodies havebeen mapped to three different regions of STH: the N-terminus, a centralregion, and the C-terminus. In FIG. 9 (top three panels), threerepresentative immunoblots, with antibodies to GST (DT12), 6XHis, andSTH (TS6), demonstrate the specificity of the monoclonal antibodies.

As depicted in FIG. 9 (bottom three panels), STH protein was detected byimmunoblots of brain homogenates from AD subjects and normal subjects,using antibodies to the N-terminus (11F11, an IgG2B), the C-terminus(10B3, an IgM), and an intervening sequence (TS6, an IgM) of STHprotein. The immunoblots show a protein with an apparent molecularweight of approximately 20 kD-6 kD more than the calculated size of 13.6kD. A similar protein is recognized by all three antibodies reactivewith three different epitopes of STH. In addition, other N-terminalantibodies to STH show a similar blotting result (data not shown). Thissize difference could be due to amino acid composition,post-translational modification, and/or aggregation with itself or otherproteins. The aggregation hypothesis would explain the presence of thehigher molecular weight bands common to all of the blots. Furtherinvestigation will determine which hypothesis (or hypotheses) is/arecorrect.

4. Conclusion

Based upon the experiments and results disclosed herein, several thingsare known about STH. STH is a nested gene in the tau locus. It has avery similar expression pattern to that of tau, suggesting that thesetwo proteins are expressed jointly, and may function together in apathway. A well-studied example of such a situation is the cholineacetyltransferase (CHAT)/vesicular acetylcholine transporter (VCHAT)gene locus. The VCHAT gene resides within the intron between exons 1 and2 of the CHAT gene. The VCHAT and CHAT genes, which are coordinatelyexpressed, are involved in the packaging of the neurotransmitter,acetylcholine (ACH), into vesicles, and in the synthesis of ACH,respectively (15). An attractive hypothesis starts to emerge that STHand tau might not only function together normally, but also may functiontogether in disease states.

A Q7R polymorphism in the STH gene was identified in human subjects, andwas found to be overrepresented in the homozygous state in the LOADpopulation. The RR genotype is suggestive of a loss-of-functionmutation, because of its homozygous state in AD patients. It is presumedthat STH has a normal cellular function. Although sequence analysis hasyielded no clues, there may be hints to the function of STH and possiblerole in AD. Since STH lacks a consensus targeting signal sequence, it isa putative cytosolic protein; thus, it may be placed in the samecompartment as tau. STH has no proline-directed phosphorylation sites,unlike tau. However, there are putative phosphorylation sites on STH forPKC and CKII-kinases that normally have significant roles in the centralnervous system and are implicated in tau phosphorylation and AD (19).Follow-up genetic studies of STH in neurodegenerative diseases with avariety of tau or amyloid pathologies could help to determine whetherSTH plays a role in the formation of neurofibrillary tangles and/oramyloid plaques.

REFERENCES

-   1. Beers and Berkow, eds., The Merck Manual of Diagnosis and    Therapy, 17^(th) ed. (Whitehouse Station, N.J.: Merck Research    Laboratories, 1999) 1395-1398, 1442-48.-   2. Myers and Goate, Curr. Opin. Neurol., 14:433, 2001.-   3. Buee et al., Brain Res. Rev., 33:95, 2000.-   4. Lewis et al., Science, 293:1487, 2001.-   5. Roks et al., Neurosci. Lett., 277:137, 1999.-   6. Crawford et al., Neurosci. Lett., 266:193, 1999.-   7. Lilius et al., Neurosci. Lett., 277:29, 1999.-   8. Baker et al., Neurosci. Lett., 285:147, 2000.-   9. Kwon et al., Neurosci. Lett., 284:77, 2000.-   10. Baker et al., Hum. Mol. Genet., 8:711, 1999.-   11. Hecker and Roux, Biotechniques, 20:478, 1996.-   12. Jicha et al., J. Neurosci., 19:7486, 1999.-   13. Ausubel et al., Current Protocols in Molecular Biology (New    York: John Wiley and Sons, New York, 1997).-   14. Altschul et al., Nucleic Acids Res., 25:3389, 1997.-   15. Y. Oda, Pathol. Int., 49:921, 1999.-   16. Gu et al., J. Neurochem., 67:1235, 1996.-   17. Saunders et al., Neurology, 43:1467, 1993.-   18. Corder et al., Cell Mol. Life Sci., 54:928, 1998.-   19. Jin and Saitoh, Drugs Aging, 6:136, 1995.-   20. Wenham et al., Lancet, 337:1158, 1991.-   21. R. Weiss, Science, 254:1292, 1991.

All publications mentioned hereinabove are hereby incorporated in theirentireties. While the foregoing invention has been described in somedetail for purposes of clarity and understanding, it will be appreciatedby one skilled in the art, from a reading of the disclosure, thatvarious changes in form and detail can be made without departing fromthe true scope of the invention in the appended claims.

1. An isolated nucleic acid sequence encoding saitohin (STH).
 2. Thenucleic acid sequence of claim 1, which is DNA, cDNA, or RNA.
 3. Thenucleic acid sequence of claim 1, which is the saitohin-Q (STH-Q)allele.
 4. The nucleic acid sequence of claim 1, comprising thenucleotide sequence set forth in FIG.
 1. 5. The nucleic acid sequence ofclaim 1, which encodes a protein comprising the amino acid sequence setforth in FIG.
 3. 6. An isolated nucleic acid sequence that hybridizesunder high stringency conditions to a second nucleic acid sequence thatis complementary to the nucleotide sequence set forth in FIG. 1 or acontiguous fragment thereof.
 7. The nucleic acid sequence of claim 1,which is the saitohin-R (STH-R) allele.
 8. The nucleic acid sequence ofclaim 1, comprising the nucleotide sequence set forth in FIG.
 2. 9. Thenucleic acid sequence of claim 1, which encodes a protein comprising theamino acid sequence set forth in FIG.
 3. 10. An isolated nucleic acidsequence that hybridizes under high stringency conditions to a secondnucleic acid sequence that is complementary to the nucleotide sequenceset forth in FIG. 2 or a contiguous fragment thereof.
 11. A purifiedsaitohin protein.
 12. The protein of claim 11, which is the saitohin-Q(STH-Q) isoform.
 13. The protein of claim 11, comprising the amino acidsequence set forth in FIG.
 3. 14. The protein of claim 11, encoded bythe nucleotide sequence set forth in FIG.
 1. 15. The protein of claim11, which is the saitohin-R (STH-R) isoform.
 16. The protein of claim11, comprising the amino acid sequence set forth in FIG.
 4. 17. Theprotein of claim 11, encoded by the nucleotide sequence set forth inFIG.
 2. 18. A purified protein encoded by a nucleic acid sequence thathybridizes under high stringency conditions to a second nucleic acidsequence that is complementary to the nucleotide sequence set forth inFIG. 1 or a contiguous fragment thereof.
 19. A purified protein encodedby a nucleic acid sequence that hybridizes under high stringencyconditions to a second nucleic acid sequence that is complementary tothe nucleotide sequence set forth in FIG. 2 or a contiguous fragmentthereof.
 20. An antibody specific for saitohin (STH) protein.
 21. Theantibody of claim 20, wherein the STH protein is the STH-Q isoform. 22.The antibody of claim 20, wherein the STH protein is the STH-R isoform.23. A method for producing an antibody specific for saitohin (STH)protein, comprising the steps of: (a) immunizing a mammal with STHprotein; and (b) purifying antibody from a tissue of the mammal or froma hybridoma made using tissue of the mammal.
 24. An antibody produced bythe method of claim
 23. 25. The method of claim 23, wherein the STHprotein is the STH-Q isoform.
 26. The method of claim 23, wherein theSTH protein is the STH-R isoform.
 27. A vector comprising a nucleic acidsequence encoding saitohin (STH).
 28. The vector of claim 27, whereinthe nucleic acid sequence is the saitohin-Q (STH-Q) allele.
 29. Thevector of claim 27, wherein the nucleic acid sequence comprises thenucleotide sequence set forth in FIG. 1 or a contiguous fragmentthereof.
 30. The vector of claim 27, wherein the nucleic acid sequencehybridizes under high stringency conditions to a nucleic acid sequencethat is complementary to the nucleotide sequence set forth in FIG. 1 ora contiguous fragment thereof.
 31. The vector of claim 27, wherein thenucleic acid sequence is the saitohin-R (STH-R) allele.
 32. The vectorof claim 27, wherein the nucleic acid sequence comprises the nucleotidesequence set forth in FIG. 2 or a contiguous fragment thereof.
 33. Thevector of claim 27, wherein the nucleic acid sequence hybridizes underhigh stringency conditions to a nucleic acid sequence that iscomplementary to the nucleotide sequence set forth in FIG. 2 or acontiguous fragment thereof.
 34. A host cell transformed with the vectorof claim
 27. 35. A host cell transformed with the vector of claim 28.36. A host cell transformed with the vector of claim
 31. 37. A method ofmaking saitohin (STH) protein, comprising the steps of: (a) introducinginto a host cell a nucleic acid sequence encoding STH; (b) maintainingthe host cell under conditions such that the nucleic acid sequence isexpressed to produce STH protein; and (c) recovering the STH protein.38. The method of claim 37, wherein the STH protein is the STH-Qisoform.
 39. The method of claim 37, wherein the STH protein is theSTH-R isoform.
 40. A transgenic non-human animal whose genome comprisesa disruption in its endogenous STH gene.
 41. The transgenic animal ofclaim 40, wherein the disruption is a knockout mutation in the STH gene.42. A transgenic non-human animal that overexpresses saitohin (STH)protein.
 43. The transgenic animal of claim 42, wherein the STH proteinis the saitohin-Q isoform.
 44. The transgenic animal of claim 42,wherein the STH protein is the saitohin-R isoform.
 45. A method fordetermining whether a subject has Alzheimer's disease (AD) or is atincreased risk for developing AD, comprising assaying a diagnosticsample of the subject for the presence of one or more alleles ofsaitohin (STH), wherein detection of the presence of STH-R allele isindicative that the subject has AD or is at increased risk fordeveloping AD.
 46. The method of claim 45, wherein the diagnostic sampleis assayed using at least one nucleic acid probe which hybridizes tonucleic acid encoding STH.
 47. The method of claim 45, wherein thenucleic acid probe is DNA or RNA.
 48. The method of claim 47, whereinthe nucleic acid probe is labeled with a detectable marker.
 49. Themethod of claim 45, wherein the diagnostic sample is assayed for thepresence of one or more alleles of STH by assaying for expression of oneor more isoforms of STH protein.
 50. The method of claim 45, wherein thediagnostic sample is assayed using an agent reactive with STH.
 51. Themethod of claim 50, wherein the agent is labeled with a detectablemarker.
 52. The method of claim 50, wherein the agent is an antibody.53. The method of claim 52, wherein the antibody is labeled with adetectable marker.
 54. A method for assessing the prognosis of a subjectwho has, or may develop, Alzheimer's disease (AD), comprising assaying adiagnostic sample of the subject for the presence of one or more allelesof saitohin (STH), wherein the presence of two STH-R alleles in thediagnostic sample of the subject indicates a more negative prognosis forthe subject.
 55. The method of claim 54, wherein the diagnostic sampleis assayed using at least one nucleic acid probe that hybridizes tonucleic acid encoding STH.
 56. The method of claim 54, wherein thediagnostic sample is assayed for the presence of one or more alleles ofSTH by assaying for expression of one or more isoforms of STH protein.57. The method of claim 54, wherein the diagnostic sample is assayedusing an agent reactive with STH.
 58. A kit for determining whether asubject has Alzheimer's disease (AD) or is at increased risk fordeveloping AD, comprising a reagent that detects the presence of one ormore alleles of saitohin (STH) and instructions for using the kit todetermine whether the subject has Alzheimer's disease (AD) or is atincreased risk for developing AD.
 59. The kit of claim 58, furthercomprising a container.